MOLECULAR STUDIES OF THE DISSOCIATION OF COMMITMENT FROM BIOCHEMICAL DIFFERENTIATION IN A VARIANT MYOBLAST CELL LINE AND THE CHARACTERIZATION OF THE RAT SARCOMERIC MYOSIN HEAVY CHAIN MULTIGENE FAMILY

Abstract

This study has focused on two major aspects. (1) The characterization of the myosin heavy chain (MHC) genes and how the structures of such genes facilitate their differential expression during muscle development. (2) The elucidation of the relationship between the two key events that lead to the terminally differentiated muscle phenotype: commitment and expression of the muscle-specific genes.;Using a cDNA clone containing embryonic MHC mRNA sequences, we have demonstrated by nucleic acid hybridization assays that the rat sarcomeric MHC is coded by a multigene family with shared sequence homology among the members. Nucleic acid hybridization and heteroduplex mapping studies of MHC genomic sequences have shown that there are coding regions which are conserved laterally and evolutionarily, that are interspersed with other coding sequences and intervening sequences that are tissue-specific. Additionally, these MHC gene sequences do not undergo rearrangement or amplification during muscle development and the embryonic MHC gene which is expressed in the L6E9 cells is transcribed as a larger nuclear RNA precursor. The study has unraveled structural similarities and differences among the tissue-specific genes which may play a role in their differential expression.;A variant myoblast cell line has been determined to be temperature-sensitive for commitment and fusion as determined from studies which include cloning assays, {lcub}3H{rcub}thymidine uptake and flow microfluorometry studies. At the temperature non-permissive for commitment, the 3b-2 cells synthesize and accumulate muscle-specific mRNAs to normal levels. Transcriptional activation of the muscle-specific genes is thus produced by processes that can be dissociated from commitment to terminal differentiation. However, in the absence of commitment, the induction of the muscle-specific genes is reversible and upon growth stimulation, the synthesis and accumulation of the muscle-specific mRNAs is decreased. The terminally differentiated muscle phenotype therefore requires the coupling of both the commitment event and the expression of the muscle-specific genes.